DESCRIPTION OF THE RELATED ART
1. Field of the Invention
The present invention relates to a thermostable diaphorase gene, a recombinant vector possessing the gene, a transformant with the recombinant vector, and a process for producing a thermostable diaphorase using the transformant.
2. Background of the Invention
Diaphorase [EC.1.6.99.-] is an enzyme functioning a significant role in the electron transport systems in vivo, and is also an industrially useful enzyme in vitro. That is, the diaphorase is an essential component for a clinical specimen in which NDA (nicotineamide adenine dinucleotide) reactions are involved. Diaphorase is currently prepared through isolation and purification from microorganisms. For example, microorganisms belonging to Clostridium (Kaplan, N. O., et al., Arch. Biochem. Biophys., Vol.132, p.91-98, 1969) and Bacillus have been known as microorganisms with diaphorase producing ability, which are commercially available from Sigma, Co. and Asahi Chemical Industry, Co., respectively. However, the yield of diaphorase obtained from these microorganisms is low and the diaphorase obtained is thermally unstable, and hence the purification of the diaphorase requires extremely laborious work.
The present inventors have found that thermophilic Bacillus stearothermophilus produces thermostable diaphorase endogenously, and have obtained the patents of the thermostable diaphorase-producing bacterium and a method for purifying the diaphorase (Japanese Patent Nos. 1715795 and 1973434).
According to these patented inventions, diaphorase with excellent thermal stability and stability under storage can be recovered and the purification thereof can be attained. However, since the thermostable diaphorase is produced by culturing the bacteria generally at a high temperature of 50 to 60.degree. C. in accordance with the patented invention described above, a vast amount of energy is required. In addition, the yield of diaphorase obtained from the bacteria is at a low level as same as conventional diaphorase. Thus, the difficulty of mass producing thermostable diaphorase has not yet been solved.